Field of the Invention
The present invention relates generally to cell culture, and more particularly to a bioreactor and method of use thereof.
Background of the Invention
The human skeleton consists of 206 distinct bones, which support and protect the body, and play a role in metabolism, calcium storage and blood cell production. Despite its ability to remodel throughout a human's lifetime and its self-healing properties, reconstructive therapies are needed to restore functionality in clinical conditions characterized by large skeletal defects resulting from congenital disorders, degenerative diseases and trauma (Braddock, M., Houston, P., et al. Born again bone: tissue engineering for bone repair. News Physiol Sci 2001, 16, 208-213). The economic burden of skeletal defects is massive and expected to rapidly increase over the next decades due to the rapid global population growth and extension of life expectancy (Hollinger, J. O., Winn, S., et al. Options for tissue engineering to address challenges of the aging skeleton. Tissue Eng 2000, 6, 341-350), with a combined annual US market for bone repair and regeneration therapies projected to reach 3.5 billion by 2017 (U.S. Markets for Orthopedic Biomaterials for Bone Repair and Regeneration. MedTech Insight 2013). A large number of bone substitute materials are currently available for skeletal reconstruction, with transplantation of bone grafts still remaining the gold standard treatment (Albert, A., Leemrijse, T., et al. Are bone autografts still necessary in 2006? A three-year retrospective study of bone grafting. Acta Orthop Belg 2006, 72, 734-740). Nevertheless, current treatments for patients in need of complex skeletal reconstruction have never reached full clinical potential and can be associated with life-threatening complications. The engineering of viable bone substitutes using a combination of patient-specific cells and compliant biomaterial scaffolds therefore represents a promising therapeutic solution.
Traditional attempts to grow bone grafts in the laboratory were based on culturing cell/scaffold constructs under static conditions in the presence of osteogenesis-inducing factors. However, static cultures are not optimal to grow centimeter-sized bone grafts for clinical translation due to poor nutrient supply and removal of metabolic waste. Under these conditions, in fact, mass transport occurs only via diffusion, which is not sufficient to support cell survival and proliferation inside the core of large cell/scaffold constructs, resulting in necrosis and poor tissue formation. In addition, cell proliferation and matrix synthesis at the construct periphery over the culture period further impede medium diffusion and contribute to the formation of a nutrient gradient that drive cell migration towards the substitute borders (Goldstein, A. S., Juarez, T. M., et al. Effect of convection on osteoblastic cell growth and function in biodegradable polymer foam scaffolds. Biomaterials 2001, 22(11), 1279-1288). On top of this, culture in static conditions does not allow provision of those biophysical stimuli that are critical for functional regeneration (Yeatts, A. B., Fisher, J. P. Bone tissue engineering bioreactors: dynamic culture and the influence of shear stress. Bone 2011, 48(2), 171-181; Klein-Nulend, J., Bakker, A. D., et al. Mechanosensation and transduction in osteocytes. Bone 2013, 54(2), 182-190). Advances in bioreactor systems over the last two decades have opened new opportunities in the field of bone engineering as they allow to nurture the development of bone tissue by providing an appropriate physiological environment with stimulatory biochemical and biophysical signals (Salter, E., Goh, B., et al. Bone tissue engineering bioreactors: a role in the clinic? Tissue Eng Part B Rev 2012, 18(1), 62-75).
Bioreactors were initially developed to allow the high-mass culture of cells used for applications in diverse areas, including fermentation, wastewater treatment and purification, food processing and drug production (Martin, I., Wendt, D., et al. The role of bioreactors in tissue engineering. Trends Biotechnol 2004, 22(2), 80-86). Many of the principles established by these applications have recently been adapted for tissue engineering purposes. A bioreactor for tissue engineering applications should (i) facilitate uniform cell distribution, (ii) provide and maintain the physiological requirements of the cell (e.g., nutrients, oxygen, growth factors), (iii) increase mass transport both by diffusion and convection using mixing systems of culture medium, (iv) expose cells to physical stimuli, and (v) enable reproducibility, control, monitoring and automation. The ultimate design of a tissue engineering bioreactor is application specific, but basic characteristics are required when engineering tissue substitutes for potential clinical applications, such as the use of materials that do not release toxic products and can withstand numerous cycle of high temperature and pressure for repeated steam sterilization in autoclaves. Furthermore, bioreactors should present a simple design in order to prevent contamination and allow quick access to the engineered tissue if any problem arises in the system during the operational period (e.g. fluid leakage and flow obstruction). Despite the fact that several design solutions and range of stress values imparted to the cells have been explored to date, bioreactors for bone engineering applications are broadly classified in few main categories, including rotating wall vessels, spinner flasks, perfusion bioreactors and compression systems (for review, see Sladkova and de Peppo (2014) Bioreactor systems for human bone tissue engineering, Processes 2(2) 494-525.).
Perfusion bioreactors for bone engineering applications are culture systems composed of several key elements, including one or more chambers where the cell/scaffold constructs are placed, a medium reservoir, a tubing circuit and a pump enabling mass transport of nutrients and oxygen throughout the perfusion chamber. Perfusion bioreactors are broadly classified into indirect or direct systems, depending on whether the culture medium is perfused around or throughout the cell/scaffold constructs.
In indirect perfusion bioreactors, the cell/scaffold constructs are loosely placed in the equilibration chamber, and the culture medium preferentially follows the path of least resistance around the constructs, resulting in reduced mass transfer throughout the core of the samples. Therefore, the convective forces generated by the perfusion pump mitigate the nutrient concentration gradients principally at the surface of the cell/scaffold constructs, thus limiting the size of bone substitutes that can be engineered using these systems. On the other hand, indirect perfusion bioreactors may represent valuable systems for the collective culture of a large number of small particulate cell/scaffold constructs that can be then assembled to repair large and geometrically complex skeletal defects (de Peppo, G. M., Sladkova, M., et al. Human embryonic stem cell-derived mesodermal progenitors display substantially increased tissue formation compared to human mesenchymal stem cells under dynamic culture conditions in a packed bed/column bioreactor. Tissue Eng Part A 2013, 19, 175-187; David, B., Bonnefont-Rousselot, D., et al. A Perfusion Bioreactor for Engineering Bone Constructs: An in Vitro and in Vivo Study. Tissue Eng Part C Methods 2011, 17(5):505-516).
In direct perfusion bioreactors, the cell/scaffold constructs are placed in the equilibration chamber in a press-fit fashion so that the culture medium is forced to pass through the center of the samples. In view of this advantage, direct perfusion bioreactors have been used to engineer bone substitutes using a combination of different human osteocompetent cells and biomaterial scaffolds (for review, see Sladkova and de Peppo (2014) Bioreactor systems for human bone tissue engineering, Processes 2(2) 494-525.). Studies demonstrate that direct perfusion of different combinations of cell/scaffold constructs highly support cell survival and proliferation, and formation of mature bone-like tissue, thus representing an optimal culture system for the construction of relevant bone substitutes with potential in clinical application of skeletal reconstructions.
While biomimetic tissue engineering strategies have been explored for ex vivo cultivation of functional bone substitutes by interfacing osteocompetent cells to biomaterials under appropriate culture conditions in bioreactors, engineering large and geometrically complex bone grafts for extensive skeletal reconstructions remains problematic using current engineering approaches. In particular, as discussed above, culture of large bone grafts is problematic using common perfusion bioreactors, due to the flow resistance caused by the large size of the graft. The development of newly formed bone tissue progressively limits the medium perfusion, with negative consequences on the functionality of the perfusion system and graft viability. Thus there remains a need for new approaches and tools to facilitate the in vitro preparation of functional bone tissue and large bone grafts. Such new approaches and tools could also be used for the in vitro preparation of other types of tissue grafts, other than bone.